Sir, Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) remain a significant challenge to public health.1 Since its first identification in The First Affiliated Hospital of Zhejiang University (FAHZU),2 KPC-2 has become the most predominant carbapenemase in China. In 2011, China detected its first four cases of NDM-1 from Acinetobacter baumannii strains. Since then, it has been recovered in most species of Enterobacteriaceae in various cities or regions of China.3 In this study, we detected and characterized a Klebsiella michiganensis isolate co-producing KPC-2, NDM-1 and NDM-5 from a patient hospitalized with acute diarrhoea in FAHZU. A patient in their early twenties presented to the bone marrow transplantation centre (BMTC) of FAHZU in December 2016. The patient received HSCT, but developed acute diarrhoea in the aftermath. Stool culture yielded a carbapenemase-producing Klebsiella isolate (designated K516). The patient was treated with trimethoprim/sulfamethoxazole (10 mg/kg/day), and the diarrhoea resolved over the next week. We subsequently did a retrospective screening of carbapenemases in carbapenem-resistant Klebsiella oxytoca cluster isolates recovered from February 2016 to August 2017. Identification of carbapenemase-encoding genes was performed as described previously.4 The clonality of K. oxytoca cluster isolates was determined via PFGE and MLST.4 Average nucleotide identity (ANI) was calculated between K516 and seven related strains (Table S1, available as Supplementary data at JAC Online). Plasmid sizes were determined using S1-nuclease PFGE (S1-PFGE) and Southern-blot hybridization.4 Conjugation experiments were performed and the resulting transconjugant strains were selected on brain heart infusion (BHI) agar plates containing 2 mg/L imipenem and 150 mg/L sodium azide. Replicon typing was conducted by multiplex PCR.5 Single-molecule real-time (SMRT) sequencing was performed and analysed as described previously.3 The nucleotide sequence data reported in this study have been submitted to the GenBank database under BioProject accession nos. PRJNA393093 and PRJNA399849. Screening of carbapenemase-producing K. oxytoca cluster isolates from the BMTC identified five blaKPC-2-positive isolates (Figure S1). K516 and K518 were both recovered from stool samples from the same patient. Interestingly, both blaKPC-2 and blaNDM were detected in isolate K516. PFGE and MLST results suggested that the polyclonal cluster of carbapenemase-producing K. oxytoca isolates existed in the BMTC (Figure S1). ANI analysis revealed that K516 is, remarkably, 100% identical to the type genome of K. michiganensis E718 (Figure S2). Therefore, K516 was identified more specifically as K. michiganensis, which belongs to the K. oxytoca cluster.6 S1-PFGE and Southern blot demonstrated that blaKPC-2 was located on an ∼126 kb plasmid that could not be typed (Figure S3). Two copies of blaNDM were detected by hybridization; these were present on an ∼106 kb IncF plasmid and an ∼46 kb IncX3 plasmid, respectively (Figure S3). The two NDM-producing plasmids were both conjugative and the transconjugants exhibited increased resistance to carbapenems (Table S2). PCR and sequencing of the transconjugants confirmed that the two blaNDM genes were blaNDM-1 and blaNDM-5, respectively. The genomic features of K516 are summarized in Table S3. The blaKPC-2-containing plasmid pK516_KPC could not be classified into any known plasmid incompatibility group. blaKPC-2 was the only determinant of antimicrobial resistance identified in pK516_KPC. Interestingly, plasmid comparison revealed that pK516_KPC was closely related to the plasmid pBKPC18-1 (GenBank accession no. CP022275), a 144 825 bp untypeable plasmid detected in a Citrobacter freundii strain from river sediment of Zhejiang Province, China (H. Xu and B. Zheng, unpublished results). The 18 kb element was missing in pK516_KPC but not in pBKPC18-1 (Figure 1a). In addition, pK516_KPC displayed 65% query coverage and 99% nucleotide identity to the plasmids pKPC2_EClY2402 (GenBank accession no. KY399972) and pKPC2_EClY2403 (GenBank accession no. KY399973) from Enterobacter cloacae, both from China. blaKPC-2 was located on a Tn3 transposon element in pK516_KPC with the linear structure klcA-korC-ΔISKpn6-blaKPC-2-ISKpn27-Tn3 (Figure 1a). This conserved structure is common among elements carrying blaKPC-2 and is widespread among Enterobacteriaceae in different regions of China.7 Figure 1 View largeDownload slide Major structural features and comparison of the three carbapenemase-producing plasmids with closely related plasmids. (a) Comparison of pK516_KPC with blaKPC-2-positive plasmid pBKPC18-1 (CP022275), pKPC2_EClY2402 (KY399972) and pKPC2_EClY2403 (KY399973). (b) Linear maps of three NDM-1-encoding plasmids, pK516_NDM1, pRJF866 (KF732966) and pKOX_NDM1 (JQ314407). (c) Schematic illustration showing the structural features of pK516_NDM5 and the closely related blaNDM-5-positive plasmids pNDM5_IncX3 (KU761328) and pM216_X3 (AP018146). Grey shading indicates shared regions with a high degree of homology. ORFs are indicated as arrows and coloured according to their putative functions. Yellow arrows indicate genes involved in conjugal transfer. Genes associated with plasmid stability are coloured orange. Blue arrows indicate replication-associated genes. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins of unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1 View largeDownload slide Major structural features and comparison of the three carbapenemase-producing plasmids with closely related plasmids. (a) Comparison of pK516_KPC with blaKPC-2-positive plasmid pBKPC18-1 (CP022275), pKPC2_EClY2402 (KY399972) and pKPC2_EClY2403 (KY399973). (b) Linear maps of three NDM-1-encoding plasmids, pK516_NDM1, pRJF866 (KF732966) and pKOX_NDM1 (JQ314407). (c) Schematic illustration showing the structural features of pK516_NDM5 and the closely related blaNDM-5-positive plasmids pNDM5_IncX3 (KU761328) and pM216_X3 (AP018146). Grey shading indicates shared regions with a high degree of homology. ORFs are indicated as arrows and coloured according to their putative functions. Yellow arrows indicate genes involved in conjugal transfer. Genes associated with plasmid stability are coloured orange. Blue arrows indicate replication-associated genes. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins of unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. The 106 140 bp plasmid pK516_NDM1 was of the IncF type. It contains two IncF replicons (IncFIB and IncFIIY), which is consistent with the structure of the previous NDM-1-producing plasmid pNDM1_EC14653 identified in China.8 Two additional antibiotic resistance genes were detected in this plasmid, i.e. rmtC and sul1 (Figure 1b). The backbone of pK516_NDM1 is identical to that of pRJF866 (GenBank accession no. KF732966), a plasmid isolated from a Klebsiella pneumoniae strain in mainland China, and to that of pKOX_NDM1 (GenBank accession no. JQ314407).9 Moreover, the genomic context of blaNDM-1 was identical to that found in various blaNDM-1-carrying plasmids in Enterobacteriaceae, which contain the conserved rmtC-ΔISAba125-blaNDM-1-bleMBL-trpF-dsbC-cutA-groES-groEL structure (Figure 1b). pK516_NDM5 was 46 149 bp in length and belonged to the IncX3 type plasmid (GC content of 46.6%). Other antibiotic resistance genes were not detected in this plasmid. BLASTN comparisons revealed that pK516_NDM5 was almost identical to plasmid pNDM5_IncX3 (GenBank accession no. KU761328)10 and pM216_X3 (GenBank accession no. AP018146), with 100% query coverage and >99% nucleotide identity. blaNDM-5 in pK516_NDM5 was preceded by Tn3, IS3000, ISAba125 and IS5D, and followed by bleMBL, trpF, dsbC and IS26 (Figure 1c). In summary, we report here what is the first case (to our knowledge) of clinical isolates producing KPC-2, NDM-1 and NDM-5. Our study further highlights the spread of carbapenemase genes in clinical settings in Zhejiang Province, including their movement into K. michiganensis. The threat of multiple carbapenemase-producing bacterial epidemics should be closely monitored, although the limitation of this study was the extremely small sample size. Acknowledgements We thank Jinru Ji and Chaoqun Ying for their assistance during sample collection and data analysis. Funding This work was supported by: the National Basic Research Program of China (No. 2015CB554201); the National Key Research and Development Program of China (No. 2016YFD0501105); the National Natural Science Foundation of China (81361138021, 81711530049, 81301461 and 41406140); the Zhejiang Provincial Key Research and Development Program (No. 2015C03032); and the Zhejiang Provincial Natural Science Foundation of China (No. LY17H190003). Transparency declarations None to declare. Supplementary data Tables S1–S3 and Figures S1–S3 are available as Supplementary data at JAC Online. References 1 Giacobbe DR, Del Bono V, Trecarichi EM et al. Risk factors for bloodstream infections due to colistin-resistant KPC-producing Klebsiella pneumoniae: results from a multicenter case-control-control study. Clin Microbiol Infect 2015; 21: 1106.e1– 8. Google Scholar CrossRef Search ADS 2 Wei ZQ, Du XX, Yu YS et al. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother 2007; 51: 763– 5. Google Scholar CrossRef Search ADS PubMed 3 Wang J, Yuan M, Chen H et al. First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4, and KPC-2 carbapenemases. Antimicrob Agents Chemother 2017; 61: e00877-17. Google Scholar CrossRef Search ADS PubMed 4 Zheng B, Zhang J, Ji J et al. Emergence of Raoultella ornithinolytica coproducing IMP-4 and KPC-2 carbapenemases in China. Antimicrob Agents Chemother 2015; 59: 7086– 9. Google Scholar CrossRef Search ADS PubMed 5 Carattoli A, Bertini A, Villa L et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219– 28. Google Scholar CrossRef Search ADS PubMed 6 Saha R, Farrance CE, Verghese B et al. Klebsiella michiganensis sp. nov., a new bacterium isolated from a tooth brush holder. Curr Microbiol 2013; 66: 72– 8. Google Scholar CrossRef Search ADS PubMed 7 Cai JC, Zhang R, Hu YY et al. Emergence of Escherichia coli sequence type 131 isolates producing KPC-2 carbapenemase in China. Antimicrob Agents Chemother 2014; 58: 1146– 52. Google Scholar CrossRef Search ADS PubMed 8 Wu W, Feng Y, Carattoli A et al. Characterization of an Enterobacter cloacae strain producing both KPC and NDM carbapenemases by whole-genome sequencing. Antimicrob Agents Chemother 2015; 59: 6625– 8. Google Scholar CrossRef Search ADS PubMed 9 Huang TW, Wang JT, Lauderdale TL et al. Complete sequences of two plasmids in a blaNDM-1-positive Klebsiella oxytoca isolate from Taiwan. Antimicrob Agents Chemother 2013; 57: 4072– 6. Google Scholar CrossRef Search ADS PubMed 10 Du H, Chen L, Tang YW et al. Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. Lancet Infect Dis 2016; 16: 287– 8. Google Scholar CrossRef Search ADS PubMed © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. 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Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Feb 1, 2018
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